Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

Abstract

Caenorhabditis elegans has 12 tRNA T,pG genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrP gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNATrpG molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressors against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNATrP gene family were differentially regulated in a tissue-or development stage-specific manner.

Uncoupling the Pleiotropic Phenotypes of clk-1 with tRNA Missense Suppressors in Caenorhabditis elegans

“…Indeed, different suppressors suppress different subsets of phenotypes, which cannot be clearly related to the amount of UQ biosynthesis they restore. Instead, the observation that they suppress different sets of phenotypes may suggest that some phenotypes have a stage-or tissue-specific focus of action and that the suppressors are stage or tissue specific as well, as are the amber suppressors (21,22,26). This would be consistent with our finding that even one copy of a particular suppressor can almost fully rescue the defecation phenotype, while even two copies of other suppressors can have no effect on this particular phenotype.…”

“…Given that it has been shown that there are important internal promoter sequences in C. elegans tRNA genes (6), it is interesting to note that all of these suppressors have identical coding sequences. This suggests the importance of additional elements outside of the coding region for regulating the expression of the various tRNA Glu genes, as has been shown for the tRNA Trp genes (21,22,26). Finally, we can speculate on why missense suppressors have not been isolated before in C. elegans.…”

Differential Expression of Individual Suppressor tRNATrp Gene Family Members In Vitro and In Vivo in the Nematode Caenorhabditis elegans

“…Trp genes displayed a hierarchy of suppression efficiencies for different amber mutations in C. elegans (19,20). The tRNAs encoded by the different family members are identical, suggesting that differences in level and/or tissue specificity of expression are responsible.…”

“…Starting recombinant plasmid clones of both wild-type and corresponding amber suppressor tRNA genes for sup -5, sup-7, sup-24, sup-28, and sup-29 were described by Kondo et al (19). Deletions of cloned suptRNA genes were prepared by the exonuclease III (exoIII)-S1 strategy of Henikoff (14).…”

“…Kondo et al (19,20) have reported on a promising system which can assay the expression of individual members of the tRNA Trp gene family of C. elegans. Genetic screens with this organism, used to characterize second-site suppressors of amber mutations, resulted in the isolation and identification of eight amber-suppressing tRNA gene mutations, all members of the tRNA Trp gene family.…”